Acid-solubilized copper-ammonium complexes and copper-zinc-ammonium complexes, compositions, preparations, methods, and uses

ABSTRACT

An antimicrobial composition is disclosed that contains an acid-solubilized copper ammonium or copper-zinc ammonium complex that is effective against microorganisms such as nosocomial or environmental bacteria, fungi, viruses, and the like. The antimicrobial composition can be used in the preparation of a medicament for treating microbes or a microbial infection, and may contain a carrier to create a cream, soap, wash, spray, dressing, cleanser, cosmetic product, topical drug product, or other antimicrobial product.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to compositions with antimicrobialactivity, and more specifically to compositions containingacid-solubilized copper-ammonium or acid-solubilizedcopper-zinc-ammonium complexes as active ingredients.

2. Description of Related Art

The antimicrobial effects of copper metal and copper salts have beenknown since ancient times. The role of copper as an anti-microbial agentwas first described in the Smith Papyrus, an Egyptian medical textwritten around 2,600 BC, which describes the application of copper tosterilize chest wounds and drinking water.

The Greeks. Romans and Aztecs used copper metal or its compounds for thetreatment of chronic infections and for hygiene in general. For example,in the Hippocratic Collection copper is recommended for the treatment ofleg ulcers associated with varicose veins. To prevent infection of freshwounds, the Greeks sprinkled a dry powder composed of copper oxide andcopper sulfate on the wound. Another antiseptic wound treatment at thetime was a boiled mixture of honey and red copper oxide. The Greeks hadeasy access to copper since the metal was readily available on theisland of Kypros (Cyprus) from which the Latin name for copper, cuprum,is derived.

The Ancient Indian ayurvedic text Charaka Samhita (300 BC) also mentionshow copper kills fatal microbes, including its role in the purificationof drinking water. Pliny (23 to 79 A.D.) described a number of remediesinvolving copper, for example, black copper oxide was given with honeyto remove intestinal worms.

In more modern times, the first observation of copper's role in theimmune system was published in 1867 when it was reported that, duringthe cholera epidemics in Paris of 1832, 1849 and 1852, copper workerswere immune to the disease. Further, animals deficient in copper havebeen shown to have increased susceptibility to bacterial pathogens suchas Salmonella and Listeria.

Copper sulfate is the key active component of the fungicidal “Bordeauxmixture” that was invented in the 19th century and is still used inagriculture today, as are numerous other copper-based agrochemicals.

Pathogenic microbes such as bacteria, fungi and viruses are responsiblefor many of the diseases in multicellular organisms as illustrated bythe following non-limiting examples.

In the pharmaceutical area, bacterial infections are involved in skindiseases such as acne (Propionibacterium acnes) and eczema(Staphylococcus aureus). Healthcare-acquired infections (HAIs) caused bymeticillin-resistant Staphylococcus aureus (MRSA), Acinetobacter sp.Klebsiella pneumonia (in which the NDM-1 enzyme gene was originallyidentified) and Legionella pneumophila which is the cause ofLegionnaire's disease. Escherichia coli (E. coli) is a common cause ofurinary tract infections. These and other HAIs are estimated to causethe death of nearly 100,000 people in the USA annually. Pathogenic E.coli 0157:117 causes gastroenteritis when ingested through contaminatedfood. Diseases caused by fungi may be relatively mild such as athlete'sfoot (Tricophyton sp.), dandruff (Malassezia globosa) and thrush(Candida albicans), but fungi such as Aspergillus fumigatus (A.fumigatus) and Candida albicans (C. albicans), and yeasts such asCryptococcus neoformans may cause life-threatening infections in immunecompromised patients. Viruses are also responsible for common diseasessuch as colds (Rhinoviruses) and influenza (Influenza viruses A. B andC) and cold sores (Herpes simplex virus), to more serious viral diseasessuch as rabies and ebola.

Bacterial infections of skin wounds such as pressure sores and diabeticleg ulcers can exacerbate the condition and copper salts andcopper-based compositions have been shown to be effective against thesediseases by virtue of both their anti-bacterial effects and theirability to stimulate wound healing by enhancing growth factorproduction.

In the cosmetic area, copper or copper-zinc compositions have been shownto be effective at, for example, ameliorating the effects of mild tomoderate sunburn and mild burns, and also in reducing the itching andinflammation caused by insect bites and reducing the appearance ofwrinkles.

In the agricultural area, fungal diseases are currently killing tanoaksin the western United States (Phytophthora ramorum) and ash trees inEurope (Chalara fraxinea). Fungal and bacterial diseases of grape vines,fruit and vegetable bearing plants and cereal crops are a threat toworld food production.

The presence of increasing numbers of drug-resistant strains ofpathogenic microbes in hospitals and in the general environment isbecoming a great concern as illustrated by the following examples.Antibiotic-resistant and multi-drug resistant strains of pathogenicbacteria such as MRSA, vancomycin-resistant Staphylococcus aureus(VRSA). Acinetobacter baumannii. E. coli and Mycobacterium tuberculosisare now commonplace. Resistant strains of pathogenic fungi such asketoconazole-resistant C. albicans and A. fumigatus are an increasingproblem particularly in immune-compromised patients. Fungicide-resistantplant pathogenic fungi constantly evolve and require new anti-fungalagents for their treatment. For example, Phytophthora infestans, thecause of potato blight, became highly resistant to Metalaxyl in the late20^(th) century. Pathogenic viruses similarly develop resistance toanti-viral drugs, the development of resistance of the H1N1 “swine flu”influenza A strain to oseltamivir phosphate (TAMIFLU®, a registeredtrademark of Genentech. San Francisco, Calif. USA) in the past fiveyears being a recent example.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided anantimicrobial composition comprising a solution comprising a copper saltin water; a basic ammonium salt added to the copper salt solution togenerate an insoluble copper-ammonium complex; and at least one watersoluble acid added to the copper salt solution to solubilize thecopper-ammonium complex and to control the pH of the clear blueacid-solubilized copper-ammonium solution thus formed.

The foregoing paragraph has been provided by way of introduction, and isnot intended to limit the scope of the invention as described in thisspecification, claims and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by reference to the following drawings,in which like numerals refer to like elements, and in which:

FIG. 1 is a graph depicting the effects of copper sulfate andcomposition Cu#1 on the growth of bacterial strain S1 (S. aureus);

FIG. 2 is a graph depicting the effects of zinc sulfate alone andcombined with composition Cu#1 on the growth of bacterial strain S1 (S.aureus);

FIG. 3 is a bar chart depicting the effects of gel, zinc sulfate (100μg/ml) alone and compositions Cu#9 or Cu#10 (200 μg/ml) alone and incombination with zinc sulfate on the growth of S1 (S. aureus);

FIG. 4 is a graph of human skin cell survival after a 24 hour culturewith copper sulfate and compositions Cu#9, Cu#10 and Cu—Zn#1: Gel+Cu#9,Gel+Cu—Zn#1; and

FIG. 5 is a graph of human skin cell survival after a 24 hour culturewith Gel+Cu#9. Gel+Cu—Zn#1, and two CLEARASIL® (a trademark of ReckittBenckiser LLC of Berkshire, England) anti-acne cream products containingthe active compounds salicylic acid and benzoyl peroxide.

The present invention will be described in connection with a preferredembodiment, however, it will be understood that there is no intent tolimit the invention to the embodiment described. On the contrary, theintent is to cover all alternatives, modifications, and equivalents asmay be included within the spirit and scope of the invention as definedby this specification, claims and the attached drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be described by way of example, and notlimitation. Modifications, improvements and additions to the inventiondescribed herein may be determined after reading this specification andviewing the accompanying drawings; such modifications, improvements, andadditions being considered included in the spirit and broad scope of thepresent invention and its various embodiments described or envisionedherein.

The inventor has surprisingly found that acid-solubilizedcopper-ammonium and copper-zinc-ammonium complexes described herein areuseful in combating specific pathogenic microbes that areantibiotic-resistant in the case of certain bacteria or otherwisedifficult to treat or control in the case of certain bacterial andfungal strains. Furthermore, the inventor has surprisingly found thatthese acid-solubilized copper-ammonium complexes can be highly effectiveagainst difficult to treat bacteria, for example, MRSA and Acinetobacterbaumannii with simultaneous lack of toxicity to human skin cells inculture at similar concentrations.

Owing to the relatively low amounts of copper that can be safelyingested (no more than 6 milligrams per day in adult humans) ortolerated by plants and animals in the environment, and particularly inthe aqueous environment, the applications of the acid-solubilizedcopper-ammonium or copper-zinc-ammonium compositions described hereinare primarily for topical use, for example, on the skins or mucousmembranes of animals and humans, on the leaves of plants, or, forexample, on surfaces in the environment, such as in homes or hospitals.

Exceptions to topical uses include, for example, (i) the treatment ofwater contaminated by one or more microbes that could be made potable bythe use of an acid-solubilized copper-ammonium or acid-solubilizedcopper-zinc-ammonium composition to remove or kill microbes, or (ii)making water fit for use by removal or killing of microalgae or bacteriacontaminating the water in a structure such as a swimming pool or hottub by the addition of one or more of the acid-solubilized copper orcopper-zinc compositions described herein.

The acid-solubilized copper-ammonium complex compositions describedherein can be applied topically to the skin of a patient for preventingor treating, for example, an MRSA infection or a fungal infection suchas C. albicans. They can also be applied topically by spraying, forexample, to the leaves of plants with a fungal infection such as powderymildew. Once on the surface, the disinfecting properties of the copperion complex can remain effective for a considerable period of time.

The acid-solubilized copper-ammonium complexes can also be applied tosurfaces, for example, either by spraying or by application with acloth, and unexpectedly can be effective against multiple differentpathogenic microbes simultaneously and can provide protection againstinfection and re-infection with such microbes. Once on the surface, thedisinfecting properties of the copper ion complex can remain effectivefor a considerable time.

The present acid-solubilized copper-ammonium complexes and substratesimpregnated therewith can provide a very effective inhibition of thegrowth of pathogenic microbes hitherto difficult to treat withconventional drugs and/or conventional disinfectant regimes. Thisinhibition of growth of pathogenic microbes can surprisingly be atconcentrations that are not toxic to human skin cells.

The acid-solubilized copper-ammonium compositions can be made intotopical formulations such as creams, gels, spray solutions, irrigationsolutions and impregnated dressings which can be for application to skinand mucosal surfaces, leaves of plants, and work surfaces in homes,hospitals, factories, vehicles etc.

The acid-solubilized copper-ammonium and copper-zinc-ammoniumcompositions are conveniently prepared according to the generalprocedure outlined below. These disclosed embodiments of the presentinvention exemplify certain preferred compositions; however, theseexamples are not intended to limit the scope of the invention. As willbe obvious to those skilled in the art, multiple variations andmodifications may be made without departing from the spirit and broadscope of the present invention.

The following example describes the general protocol for makingacid-solubilized copper-ammonium complex formulation Cu#3. All chemicalswere obtained from Sigma-Aldrich Company Ltd., The old Brickyard, NewRoad, Gillingham. Dorset SP8 4XT, UK, or Oxoid Ltd., Wade Road.Basingstoke, Hampshire RG24 8PW, UK.

16.0 grams of copper sulfate pentahydrate was added to 80 milliliters ofdistilled water in a glass beaker using a magnetic stirrer and a stirbar with vigorous mixing to form a clear blue solution. 4.0 grams ofammonium carbonate was added to the copper sulfate solution in 0.5-1gram amounts owing to the vigorous evolution of carbon dioxide which wasallowed to subside between additions of the aliquots of ammoniumcarbonate. A pale blue insoluble copper-ammonium complex was formedduring the addition of the ammonium carbonate to the copper sulfatesolution and this was kept in suspension with vigorous stirring. 10.0milliliters of phosphoric acid (85% solution) was gradually added withthe evolution of carbon dioxide and the solubilization of thecopper-ammonium complex. Sufficient acid must be added so as tocompletely solubilize the copper-ammonium complex and to control the pHof the solution. The resulting clear, brilliant blue solution wasvigorously stirred for a further 5 minutes and then made up to a finalvolume of 100 milliliters with distilled water.

It is preferred that the elemental concentration of copper in thecompositions is of the order 1 to 10 grams/deciliter preferably 3 to 7grams/deciliter, more preferably 3.5 to 5 grams/deciliter, with thesolvent phase being distilled or deionised water.

For examples of other acid-solubilized copper-ammonium complexformulations based on this protocol see Table 1 below. In the Table,gram or grams is abbreviated with the letter g and milliliter ormilliliters being abbreviated with the letters ml.

TABLE 1 Examples of acid solubilized copper- ammonium complexcompositions (Cu#) Ingredients added per deciliter of distilled waterComposition CuSO₄•5H₂0 (NH₄)₂CO₃ Acid Cu#1  16.0 g* 2.0 g 5.0 g H₃PO₄(85%) Cu#3 16.0 g 4.0 g 10.0 g H₃PO₄ (85%) Cu#3a 16.0 g NH₄OH (10% 2.0 gH₃PO₄ (85%) solution) 3.2 ml Cu#9 16.0 g 2.0 g 2.3 ml HCl (~18M) Cu#1016.0 g 2.0 g 1.5 ml Glacial acetic acid Cu#10a 16.0 g NH₄OH (10% 1.5 mlGlacial solution) 1.6 ml acetic acid Cu#11a 16.0 g NH₄OH (10% 1.5 gCitric acid solution) 1.6 ml Cu#12 16.0 g 1.5 g 1.0 ml H₂SO₄ (37%) Cu#13CuCl₂•2H₂O 3.0 g 2 ml H₂SO₄ (37%) 10.7 g* Cu#16  15.0 g** NH₄HCO₃ 6.0 g8.0 g H₃PO₃ Cu#27 15.0 NH₄OH (10% 2.4 g H₃PO₃ solution) 2.0 ml *Theindicated amounts produce a stock solution of approximately 4.0grams/deciliter or **3.75 grams/deciliter of elemental copper.

The following example describes the general protocol for makingacid-solubilized copper-zinc-ammonium formulation Cu—Zn#10.

6.0 grams of copper sulfate pentahydrate was added to 80 milliliters ofdistilled water in a glass beaker using a magnetic stirrer and a stirbar with vigorous mixing to form a clear blue solution. 6.6 grams ofzinc sulfate heptahydrate was added to the copper sulfate solution andstirred until dissolved. 2.0 milliliters of ammonium hydroxide (10%solution) was added to the copper sulfate/zinc sulfate solutiondropwise. A pale blue insoluble copper-zinc-ammonium complex was formedduring the addition of the ammonium hydroxide solution to the coppersulfate/zinc sulfate solution and this was kept in suspension withvigorous stirring. 4.0 grams of phosphorous acid was gradually added inapproximately 0.5 g aliquots to solubilize the copper-zinc-ammoniumcomplex. Sufficient acid must be added so as to completely solubilizethe copper-zinc-ammonium complex and to control the pH of the solution.The resulting clear, pale blue solution was vigorously stirred for afurther 5 minutes and then made up to a final volume of 100 milliliterswith distilled water.

It is preferred that the elemental concentration of copper and zinc inthe compositions is of the order 1:1 with the solvent phase beingdistilled or deionised water.

For other acid-solubilized copper-zinc-ammonium formulations based onthis protocol see Table 2. In the Table, gram or grams is abbreviatedwith the letter g and milliliters is abbreviated as ml.

TABLE 2 Examples of acid-solubilized copper-zinc compositions (Cu—Zn#)Compo- Ingredients added per dL of distilled water sition CuSO₄•5H₂0ZnSO₄•7H₂O (NH₄)₂CO₃ Acid Cu—Zn#1  6.0 g*  6.6 g* 2.0 g 3.0 ml HCl(~18M) Cu—Zn#2 6.0 g 2.2 g 2.0 g 3.0 ml HCl (~18M) Cu—Zn#3 6.0 g 1.1 g2.0 g 3.0 ml HCl (~18M) Cu—Zn#4 2.0 g 6.6 g 2.0 g 3.0 ml HCl (~18M)Cu—Zn#5 1.0 g 6.6 g 2.0 g 3.0 ml HCl (~18M) Cu—Zn#7 6.0 g 6.6 g 2.0 g5.0 g H₃PO₃ Cu—Zn#10 6.0 g 6.6 g NH₄OH 5.0 g H₃PO₃ (10% solu- tion) 2.0ml *The indicated amounts produce a stock solution of approximately 1.5grams/deciliter of both elemental copper and elemental zinc.

Methods for making Aloe vera-based gel of the present invention aredescribed herein, with examples provided.

Example 1

Sprinkle 1.0 gram of xanthan gum onto 150 milliliters of distilled waterat around 60° C. in a 300 milliliter borosilicate glass beaker and mixvigorously with a whisk for 30 seconds to form a smooth, bubblysolution. Sprinkle 1.0 gram of Aloe vera powder onto the xanthan gumsolution and whisk vigorously again for 30 seconds or until the Aloevera powder is fully dissolved into the solution. Add room temperaturedistilled water to a final is volume of 200 milliliters. Store the gelat 4° C. and use within one month. This gel was used for diluting thecopper and copper/zinc compositions in the agar plate antibacterialtests. If the gel is to be used for cosmetic purposes and/or storage atroom temperature then antimicrobial preservatives must be added. Thesemay include, for example, mandelic acid, glycerol, potassium sorbate,phenoxyethanol, sodium benzoate. An effective combination is potassiumsorbate (KS) and sodium benzoate (NaB). Separate stock solutions of10.0% weight/volume of KS and NaB were prepared and 2.0 milliliters ofeach product was added to the xanthan gum-Aloe vera solution describedabove with vigorous mixing and then room temperature distilled water wasadded to a final volume of 200 milliliters. Acid-solubilizedcopper-ammonium or copper-zinc-ammonium complex compositions werediluted, for example, 1:100 vol/vol into this gel.

Example 2

In a weighed beaker gradually mix 3.0 grams of xanthan gum in −1 gramaliquots into 3 grams of glycerol at room temperature (around 20° C.)until a smooth homogenous paste is formed. Re-weigh the beaker and itscontents. Gradually add 5 grams of the glycerol-xanthan gum mixture (1gram of the mixture should remain in the beaker) to 400 milliliters ofdistilled water at around 60° C. in a 800 milliliter borosilicate glassbeaker whilst vigorously whisking until a clear, bubbly solution isformed. Sprinkle 2.5 grams of Aloe vera powder onto the solution andcontinue whisking until the Aloe vera powder is fully dissolved into thesolution. Antimicrobial preservatives (e.g. 5.0 milliliters of the 10%/ostock solutions of KS and NaB as described above) can be added at thisstage with whisking before adding room temperature distilled water to afinal volume of 500 milliliters.

In order that the present invention may be illustrated, more easilyappreciated and readily carried into effect by those skilled in the art,embodiments thereof will now be presented by way of non-limitingexamples only and described with reference to the accompanying drawings.

Example 1 Agar Plate Test for the Antibacterial Effect of SelectedCompositions on Bacteria

The Staphylococcus aureus strain (S1) isolated from the skin of aneczema patient was cultured on tryptone-soya agar (TSA) and a singlecolony was cultured in RPMI-1640 medium at 37° C. overnight. The S1 (S.aureus) culture was diluted 1:10 with fresh RPMI-1640 medium and 0.2milliliter was spread onto a 9 centimeter Petri dish containing TSA andallowed to dry. The Propionibacterium acnes strain ATCC 11828 (P. acnes)was cultured in brain-heart infusion broth with 1% glucose (BHI) at 37′Cfor 72 hours under anaerobic conditions using an AnaeroGen sachet. A 0.2milliliter sample of the P. acnes culture was spread onto a 9 centimeterPetri dish containing BHI agar and allowed to dry.

The composition to be tested was either diluted with distilled water and5 microliters was placed onto a 5 millimeter filter paper disc or wasadded to the Aloe vera-based gel (without preservatives) described aboveat various concentrations and a 5 microliter drop was placed onto theagar surface. The plates were then incubated at 37° C. for 24 hours(under anaerobic conditions for P. acnes), when the zones of inhibition(ZoI) were measured in millimeters) twice at a 900 angle using a rulerand the average diameter (D) was calculated. The area of inhibition inmm² (AoI) was calculated using the formula: AoI=π×(D/2)².

Results:

1. The results in FIG. 1 compare the effects of copper sulfate andcomposition Cu#1 dissolved in gel on the growth of bacterial strain S1(S. aureus) on TSA. It is clear that composition Cu#1 is approximately4-fold more potent than copper sulfate at inhibiting the growth of S1(S. aureus), since copper sulfate at 800 micrograms/milliliter andcomposition Cu#1 at 200 micrograms/milliliter have the same AoI (28mm²). The results show that compositions of acid-solubilizedcopper-ammonium complexes are surprisingly more antimicrobial thancopper sulfate alone.2. The results in FIG. 2 compare the effects of zinc sulfate alone andcombined with composition Cu#1 dissolved in gel on the growth ofbacterial strain S1 (S. aureus) on TSA. Zinc sulfate alone had no effecton the growth of S1 (S. aureus) at any concentration tested (50-200micrograms/milliliter). In contrast, when composition Cu#1 (100micrograms/milliliter) was combined with increasing concentrations ofzinc sulfate there was a clear synergistic inhibitory effect on thegrowth of S1 (S. aureus). Thus, composition Cu#1 alone had an AoI of 3mm², but this increased to 13, 20 and 28 mm² when combined with zincsulfate at concentrations of 50, 100 and 200 micrograms/milliliter,respectively. These results surprisingly show that antimicrobialacid-solubilized copper-ammonium complexes can act synergistically withzinc sulfate which alone has no antimicrobial activity against strain S1(S. aureus). These results suggest that acid-solubilizedcopper-zinc-ammonium complexes may have advantageous antimicrobialactivity against certain organisms.3. FIG. 3 shows the effect of two compositions, Cu#9 and Cu#10 (200micrograms/milliliter) in gel, alone and combined with zinc sulfate (100micrograms/milliliter) in gel on the growth of S1 (S. aureus). Theresults show that the gel alone and zinc sulfate in gel have no effecton the growth of S1 (S. aureus). Compositions Cu#9 and Cu#10 in gelalone both inhibited the growth of S1 (S. aureus) with AoIs of 20 and 33mm², respectively. In contrast, when combined with zinc sulfate in gel,compositions Cu#9 and Cu#10 showed increased inhibition of the growth ofS1 (S. aureus) with AoIs of 38 and 57 mm², respectively. These resultsconfirm and extend those shown in FIG. 2, demonstrating that there is aclear synergistic inhibitory effect on the growth of strain S1 (S.aureus) between acid-solubilized copper-ammonium complexes and zincsulfate.4. Table 3 shows the effect of copper sulfate, five differentacid-solubilized copper-ammonium complexes and one acid-solubilizedcopper-zinc-ammonium complex on the growth of P. acnes on 13BI agar asdescribed in the methods section above. All of the acid-solubilizedcopper-ammonium complexes stock solutions were diluted 1:200 (vol/vol)and a copper sulfate solution of the same concentration of elementalcopper (200 microgram/milliliter) was also prepared. The stock solutionof composition Cu—Zn#1 (see Table 2) was also diluted 1:200 for testing.All dilutions were in distilled water.

TABLE 3 The effect of copper sulfate and various compositions (describedin Tables 1 and 2) on the growth of P. acnes on BHI agar. There was nocorrelation between the antibacterial activity and the pH of thecompositions. Composition Zone of Inhibition (mm) pH of stock solutionsCopper sulfate 13 3.4 Cu#3 16 1.6 Cu#9 16 2.2 Cu#10 18 4.4 Cu#12 19 2.0Cu#13 16 1.0 Cu—Zn#1 18 1.5

The results in Table 3 show that all of the acid-solubilizedcopper-ammonium complexes were more inhibitory to the growth of P. acnesthan an equivalent concentration of elemental copper in the form ofcopper sulfate. Although the ZoIs for the five Cu# compositions testedwere similar, the formulations of the compositions were quitedifferent—as shown in Table 1. Cu#3. Cu#9 and Cu#13 had the same ZoI butdiffered in the acid used to solubilize the copper-ammonium complex(phosphoric, hydrochloric and sulfuric acids, respectively) and Cu#13comprises copper chloride rather than copper sulfate. Cu#12 (coppersulfate and sulfuric acid) had the greatest ZoI against P. acnes butdiffers only in the copper salt used in the composition from Cu#13 (seeTable 1).

Since the copper-ammonium and copper-zinc-ammonium compositions aresolubilized with acids it could be suggested that the acidity of thecompositions is responsible for their greater antimicrobial activitywhen compared to copper sulfate. However, as shown in Table 3, the stocksolution of Cu#10 has a higher pH than an equivalent solution of coppersulfate and yet has a greater inhibitory effect on the growth of P.acnes. Furthermore, a non-parametric Spearman correlation test betweenthe ZoIs and pH's of the compound/compositions shown in Table 3 yieldeda correlation coefficient=−0.056 with p=0.81 (using the Prism 6statistics package by GraphPad Software in La Jolla, Calif.), showingthat there is no significant correlation between pH and antimicrobialactivity for the compositions tested in this experiment.

Interestingly, composition Cu—Zn#1 was one of the more inhibitorycompositions despite having only 37.5% of the elemental copper of theCu# compositions, but in conjunction with an equimolar concentration ofelemental zinc (1.5 grams/deciliter in the stock solution). Theimpressive activity of Cu—Zn#1 supports the conclusions from the resultspresented in FIGS. 2 and 3, that there is synergy between copper andzinc ions and shows that this acid-solubilized copper-zinc-ammoniumcomplex demonstrates a greater than expected inhibitory effect againstP. acnes. These results also suggest that composition Cu—Zn#1 may havetherapeutic value in the treatment of acne, a disease stronglyassociated with P. acnes. Furthermore, since it is known that P. acnesdoes not grow well at pH 4.5-5, which is the pH range of “normal” skin,the acid-solubilized copper-zinc-ammonium complex Cu—Zn#1 may be oftherapeutic value in the treatment of acne since it strongly inhibitsthe growth of P. acnes and the pH values of a 1% (vol/vol) formulationof Cu—Zn#1 in the gel with preservatives is around pH 4.6.

Example 2 Bacterial Microplate Cultures

The aerobic bacterial strains Acinetobacter baumannii (A. baumannii),methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiellapneumonia (K. pneumoniae) were isolated from swabs taken in a hospitalward. Escherichia coli (NCTC 9001; E. coli) is a clinical isolate from apatient's urinary bladder; all strains were cultured in RPMI-1640. Stocksolutions of the compounds/compositions to be tested were diluted sothat double dilutions in the microplate assays commenced at a 1% vol/volconcentration. Double dilutions of the compositions to be tested wereprepared in RPMI-1640 in a 96-well tissue culture plate. The bacterialcultures were diluted 1:40 with fresh RPMI and added to thecompositions. P. acnes was cultured as described in EXAMPLE 1 (Agarplate test methods) and a sample was diluted 1:100 with fresh BHI.Double dilutions of the compositions to be tested were prepared asdescribed above in distilled water in a 96-well tissue culture plate(100 microliters) and 100 microliters of the diluted P. acnes culturewas added. The plates were then incubated at 37° C. for 24 hours (underanaerobic conditions for P. acnes). The minimum inhibitory concentration(MIC) of the compositions was defined as the lowest concentration atwhich no visible bacterial growth was observed.

Results

1. Table 4 shows the MIC results for copper sulfate, zinc sulfate andvarious compositions described in Tables 1 and 2. In Experiment #1, thetwo copper-based compositions tested (Cu#3 and Cu#9) were twice asactive as copper sulfate. Surprisingly, zinc sulfate had a lower MICwith P. acnes than any of the Cu-based products and the acid-solubilizedcopper-zinc-ammonium formulation Cu—Zn#1 was the most active compositiontested. Cu—Zn#3 which has the same amount of elemental copper as Cu—Zn#1but 6-fold less elemental zinc (see Table 2) was 4-fold less active thanCu—Zn#1.

TABLE 4 The effect of copper sulfate and zinc sulfate and variouscompositions (described in Tables 1 and 2) on the growth of P. acnes inbacterial microplate cultures. Experiment #1 Experiment #2 CompositionMIC (μg/ml) Composition MIC (μg/ml) Copper sulfate 100 Copper sulfate200 Cu#3 50 Cu#3 100 Cu#9 50 Cu#12 100 Zinc sulfate 25 Cu#13 100 Cu—Zn#112.5 Cu—Zn#1 12.5 Cu—Zn#3 50 Cu—Zn#3 50In Experiment #2, the MICs for copper sulfate and Cu#3 with P. acneswere double those seen in the first experiment but Cu#3 was still twiceas active. Cu#12 and Cu#13 had the same MIC as Cu#3 despite comprisingsulfuric acid rather than the phosphoric acid in Cu#3; Cu#13 comprisedcopper chloride rather than copper sulfate in Cu#3 and Cu#12. Theseresults indicate that the acid-solubilized copper-ammonium complexes aregenerally more active than copper sulfate alone. As in Experiment #1,Cu—Zn#1 was the most active composition against the growth of P. acnesand was again 4-fold more active than Cu—Zn#3.

Overall these experiments confirm and extend the agar plate test resultsdescribed above and shown in Table 3. The results confirm thatacid-solubilized copper-ammonium compositions are more active thancopper sulfate alone. The present results surprisingly showed that zincsulfate inhibits the growth of P. acnes and that Cu—Zn#1 wasconsiderably more active than any of the Cu# compositions, despitecontaining only 37.5% of the elemental copper. The results show that thecombination of equimolar elemental zinc and copper in compositionCu—Zn#1 is the more active than zinc sulfate alone and even more activethan Cu—Zn#3 which contains 6-fold less elemental zinc compared tocopper. Overall the results of these experiments confirm that Cu—Zn#1may have therapeutic benefit in acne.

2. Table 5 shows the MIC results for Cu—Zn compositions #1 to #5 whichhave varying ratios of elemental copper and zinc, whilst the amounts ofammonium carbonate used to make the Cu—Zn complexes and the amount ofhydrochloric acid used to solubilize the Cu—Zn complexes remainedconstant (see Table 2). In this experiment, composition Cu#3 (used as areference between experiments) had an MIC=50 micrograms/milliliter asdid zinc sulfate. As in the previous experiments, composition Cu—Zn#1was 4-fold more active against the growth of P. acnes than Cu—Zn#3 andin this experiment Cu—Zn#2. Cu—Zn#4 and #5 were both equally half asactive as Cu—Zn#2 and #3 and 8-fold less active than Cu—Zn#1. Theseresults show that composition Cu—Zn#1 comprising equimolar elementalcopper and zinc is the most active composition against the growth of P.acnes of all those tested. The results with Cu—Zn#2 and #3 suggest thatthe role of copper against the growth of P. acnes is somewhat greaterthan that of zinc, but the surprising synergy of the two metals togetherwhen in equimolar equivalence (Cu—Zn#1) creates by far the mosteffective composition against the growth of P. acnes.

TABLE 5 The effect of various Cu—Zn# compositions, Cu#3 and zinc sulfateon the growth of P. acnes in bacterial microplate cultures. CompositionRatio of elemental Cu:Zn MIC (μg/ml) Cu#3 — 50 Zinc sulfate — 50 Cu—Zn#11:1   6.3 Cu—Zn#2 1:0.33 25 Cu—Zn#3 1:0.17 25 Cu—Zn#4 0.33:1     50Cu—Zn#5 0.17:1     503. Table 6 shows the MIC results for copper sulfate and various Cu# andCu—Zn# compositions against the growth of MRSA, E. coli, A. baumanniiand K. pneumoniae in microplate cultures. MRSA and A. baumannii are bothclearly more sensitive to the effects of the copper sulfate, Cu# andCu—Zn# compositions than E. coli and K. pneumonia, with A. baumanniibeing surprisingly sensitive to the inhibitory effect of the Cu—Zn#compositions. Since A. baumannii is a particularly persistent bacteriumthat causes healthcare acquired infections (HAIs), cleaning and/orfogging with composition Cu—Zn#1 may be particularly useful in freeinghospitals and other healthcare facilities from this increasinglyprevalent and difficult to treat microbe.

TABLE 6 The effect of copper sulfate and various Cu# and Cu—Zn#compositions (described in Tables 1 and 2) on the growth, of MRSA, E.coli, A. baumannii and K. pneumoniae in bacterial microplate cultures.Composition MRSA E. coli A. baumannii K. pneumoniae Copper sulfate 12.5100 6.3 50 Cu#3 6.3 50 3.1 12.5 Cu#9 6.3 50 3.1 25 Cu#10 6.3 100 3.1 25Cu#12 6.3 50 1.6 25 Cu#13 6.3 100 3.1 25 Cu—Zn#1 25 200 0.31 3.1 Cu—Zn#325 200 0.63 6.3 Cu#3a 6.3 50 1.6 NT Cu10a 6.3 50 1.6 NT Cu#11a 6.3 501.6 NT NT = Not tested.

MRSA and E. coli were both more sensitive to the growth inhibitoryeffects of the Cu# compositions than the Cu—Zn# compositions and coppersulfate, whilst the opposite was true for A. baumannii and K.pneumoniae. However. E. coli was generally around 8-fold less sensitiveto all of the compositions tested than MRSA.

It was noted that certain compositions made with ammonium carbonate orammonium hydrogen carbonate and using acetic acid (Cu#10) and citricacid (Cu#11: not shown in Table 1 or tested because precipitation wasnoticeable after 24 hr), started to precipitate within days ofpreparation. Very similar compositions were made using ammoniumhydroxide and these compositions (Cu#10a and Cu#11a) proved to besurprisingly more stable and showed no signs of precipitation aftermonths of preparation (storage at room temperature). Three compositionswere made using ammonium hydroxide (see Table 1 for details) and testedagainst MRSA, A. baumannii and E. coli as shown at the bottom of Table6. Cu#3 is a stable composition and Cu#3a was equally as stable andactive against the three bacterial strains tested. Cu10a showed similaractivity to Cu#10 and Cu#11a with citric acid was as active as the otherCu# compositions. Not only are these (and other compositions preparedwith ammonium hydroxide—see below) compositions more stable, they arequicker and easier to make because no carbon dioxide is liberated, andthe Cu—/Cu—Zn-ammonium complexes formed with ammonium hydroxide requireless acid for solubilization, so the pH of the compositions can behigher, representing advantages in formulations thereof.

MRSA is perhaps the best known and most problematic pathogenic bacteriumcausing HAIs, but the USA300 MRSA strain has also become an increasingconcern in community-associated infections particularly of the skin.Carbapenem-resistant K. pneumoniae (CRKP) has emerged over the past 10years to become a worldwide nosocomial pathogen that is almost totallydrug-resistant. However, the sensitivity of the bacterial strains testedto Cu# and Cu—Zn# compositions Table 6), suggests that thesecompositions may be useful for cleaning, fogging and the like inhospitals, homes and other buildings to remove these organisms. Overall,since A. baumannii is also rather sensitive to the Cu# compositions,Cu#3 may be a particularly effective composition for cleaning anddisinfection to remove these pathogenic microbes from buildings.

Example 3 Cytotoxicity Assay with a Human Squamous Epithelial Skin CellLine (A-431)

A-431 (ATCC) was cultured in RPMI-1640 medium supplemented with 10%fetal bovine serum and 2 mM L-glutamine (complete medium) in ahumidified incubator at 37′C with a 5% CO₂ in air atmosphere. Forcytotoxicity experiments the cells were trypsinized, washed, counted and2×10⁴ cells were plated into the wells of flat-bottom 96-well plates incomplete medium and cultured for 3 days until a confluent monolayer wasformed. The products tested were: (i) Copper sulfate, Cu#9, Cu#10 andCu—Zn#1 as shown in FIG. 4, and (ii) compositions Cu#9 and Cu—Zn#1diluted in the Aloe vera gel with preservatives along with twocommercial acne creams: CLEARASIL® (a registered trademark of ReckittBenckiser LLC of Berkshire England) ultra rapid action treatment gelwith 2% salicylic acid and CLEARASIL® (a registered trademark of ReckittBenckiser LLC of Berkshire England) daily clear acne treatment creamwith 10% benzoyl peroxide as shown in FIG. 5. All test products werediluted in complete medium and then added in triplicate to the confluentA-431 cells which were then cultured for a further 24 hours whencytotoxicity/cell survival was quantitatively assessed using thesulforhodamine B (SRB) assay as described below.

The cells were washed twice with RPMI-1640 medium and then fixed with10% trichloroacetic acid for 1 hour at 4° C. After washing twice withtap water, the cells were stained with SRB (0.4% w/v SRB in 1% aceticacid) for 30 min at room temperature. After washing twice with tap waterthe remaining stain was dissolved in 10 mM Tris base and the absorbanceof the wells was measured on a Dynatech Multiplate ELISA reader at 540nm. Percent cell survival was calculated by dividing the test productabsorbance by control absorbance and multiplying by 100.

Results

FIG. 4 shows that copper sulfate and compositions Cu#9 and #10 hadalmost identical cytotoxicity profiles with human skin cells with 50%cytotoxic concentrations (CC₅₀) of 50 micrograms/milliliter, suggestingthat copper ions are responsible for the cytotoxic effects of the threeproducts. The composition Cu—Zn#1 was slightly more cytotoxic than theother three products (CC₅₀=30 micrograms/milliliter), presumably owingto the presence of both copper and zinc ions, albeit with the elementalcopper concentrations being only 37.5% of the other three products,suggesting that zinc ions are even more cytotoxic than copper ions tohuman skin cells and/or they enhance the cytotoxicity of copper ions.

Importantly, these results show that compositions described herein suchas Cu#3, #3a, #9, #10, #10a, #11a, #12, #13 and Cu—Zn#1 and #3 caninhibit the growth of bacteria such as MRSA and A. baumannii atconcentrations (MIC 1.6-6.3 microgram/milliliter—see Table 6) well belowthose that cause cytotoxicity to human skin cells in culture (around 30micrograms/milliliter). Cu—Zn#1 also inhibits the growth of P. acneswith an MIC=12.5 micrograms/milliliter (Table 4) at which concentrationit is not cytotoxic to human skin cells.

FIG. 5 shows the effects of gel containing 1% vol/vol of compositionsCu#9 (400 micrograms/milliliter elemental copper) and Cu—Zn#1 (150micrograms/liter elemental copper and zinc) and two commerciallyavailable CLEARASIL® (a registered trademark of Reckitt Benckiser LLC ofBerkshire England) acne creams on the survival of human skin cells intissue culture. Both of the CLEARASIL® (a registered trademark ofReckitt Benckiser LLC of Berkshire England) products, one containing 10%benzoyl peroxide and the other containing 2% salicylic acid as activeingredients, produced similar cytotoxicity profiles with CC₅₀ values of0.02% and 0.05%, respectively, and 100% cytotoxicity (0% cell survival)at concentrations around 0.5% and above. Gel containing 1% ofcomposition Cu—Zn#1, which would be the preferred composition fortreating acne, had a CC₅₀ of 4%, while the gel containing 1% ofcomposition Cu#9 did not achieve a CC₅₀ even at 10% vol/vol in theassay. These gel formulation CC₅₀ values are compatible with the CC₅₀values obtained with the compositions alone (see FIG. 4) Therefore, itis clear that the CLEARASIL® (a registered trademark of ReckittBenckiser LLC of Berkshire England) products are around 200-fold moretoxic to human skin cells than either of the gel formulations.

It is important to note that owing to the relative simplicity of skincell cultures compared to the complex structure of skin, the healthcareimplications need to be interpreted with care and these in vitro resultsmay not translate into clinical differences. Nevertheless, it appearsthat the active ingredients in the CLEARASIL® (a registered trademark ofReckitt Benckiser LLC of Berkshire England) products, salicylic acid andbenzoyl peroxide, which are widely used in acne treatment products, areparticularly toxic to skin cells which may be more exposed on the skinof people suffering from acne. Thus the gel with the Cu—Zn#1 formulationmay be less damaging to the skin of acne sufferers, whilst being highlyactive against the P. acnes, the bacterium associated with acne (seeEXAMPLES 1 and 2).

Example 4 Anti-Fungal Assays with Plant Pathogenic Fungi

Chalara fraxinea isolate 196/28 (C. fraxinea), Aspergillus niger (A.niger) and a strain isolated from powdery mildew (Pm; isolated from anapple leaf) were cultured on potato dextrose agar (PDA) at roomtemperature (around 22° C.). To assess the effects of compositions onfungal growth, 10 microliters of the test compositions diluted insterile distilled water were placed in the wells of a 12-well tissueculture plate and 1 milliliter of PDA was added by pipette to each well.The plate was agitated to evenly distribute the test composition evenlythrough the agar (distilled water alone was used as control) and thenthe agar was allowed to set. Plugs of agar containing fungal hyphae (3mm²) from an established fungal culture were carefully cut out using ascalpel and inserted into holes in the centre of the agar in each wellof the 12-well plate which was then cultured at room temperature (around22° C.). Since the rate of fungal growth varies depending on the strain,the experiments were assessed after 3 days for Pm, and at appropriateintervals of 3-4 days for A. niger and around 7 days for C. fraxinea. Toassess the effects of compositions on fungal growth, the diameter of thefungal hyphae was measured twice at a 90° angle using a ruler and theaverage diameter in millimeters was calculated. If no fungal growthcould be detected by eye, the cultures were observed by phase microscopy(40×) to confirm that there was no growth (NG) as judged by visualexamination.

Results

1. Table 7 shows the effects of copper sulfate and composition Cu#16 onthe growth of C. fraxinea. Composition Cu#16 strongly inhibited fungalgrowth at a concentration of 125 micrograms/milliliter at both timepoints and was around 30-times more inhibitory to fungal growth thancopper sulfate (results with 3.75 micrograms/milliliter Cu#16 were verysimilar to those with 125 micrograms/milliliter of copper sulfate),presumably owing to the solubilization of the copper-ammonium complex inCu#16 with phosphorous acid (see Table 1), which is known to haveanti-fungal activity.

TABLE 7 The effects of copper sulfate and composition Cu#16 on thegrowth of C. fraxinea on PDA. Diameter of fungal growth (mm) Day 7 Day15 CuSO₄ (μg/ml) 125 10 18 37.5 13  20** 12.5 14 20 3.75 14 20 Cu#16(μg/ml) 125  3*  4 37.5  7 10 12.5  9 15 3.75 10 17 Control 14 20*Slight growth observed by phase microscopy at 40X magnification. **20millimeters is the width of the wells and represents confluent fungalgrowth.2. Table 8 shows the effects of compositions Cu—Zn#1 and Cu—Zn#7 on thegrowth of C. fraxinea. Composition Cu—Zn#1 is less effective thancomposition Cu—Zn#7 at inhibiting the growth of C. fraxinea and althoughthe formulations of the two compositions are very similar (Table 2),composition Cu—Zn#7 contains phosphorous acid as the copper-zincsolubilizing acid whereas Cu—Zn#1 contains hydrochloric acid.Phosphorous acid is known to have anti-fungal activity and its use incomposition Cu—Zn#7 results in an anti-fungal composition that is 3- to10-fold more active than Cu—Zn#1 on days 7 and 14 of the assay,respectively. A 1% concentration of Cu—Zn#7 contains 150micrograms/milliliter of elemental copper, so comparing the effects ofCu—Zn#7 to those of copper sulfate and Cu#16 in Table 7 indicates thatCu—Zn#7 is a slightly less effective inhibitor of C. fraxinea growththan Cu#16, but it is considerably more effective than copper sulfate.

TABLE 8 The effect of compositions Cu—Zn#1 and Cu—Zn#7 on the growth ofC. fraxinea on PDA. The Cu—Zn# compositions were used as a percentdilution of the stock solutions (Table 2), so a 1% solution contains 150μg/ml of elemental copper (and zinc). Diameter of fungal growth (mm) Day7 Day 14 Cu—Zn#1 (%) 1 7 18 0.3 16  20* 0.1 16 20 0.03 16 20 0.01 16 20Cu—Zn#7 (%) 1 4  8 0.3 8 14 0.1 15 17 0.03 16 19 0.1 16 20 Control 16 20*20 millimeters is the width of the wells and represents confluentfungal growth.3. Table 9 shows the effects of copper sulfate and composition Cu#16 onthe growth of A. niger. Composition Cu#16 completely inhibited fungalgrowth at a concentration of 125 micrograms/milliliter and at all threetime points was 3- to 10-fold more inhibitory to fungal growth thancopper sulfate as was seen with C. fraxinea (Table 7).

TABLE 9 The effects of copper sulfate and composition Cu#16 on thegrowth of A. niger on PDA. Diameter of fungal growth (mm) Day 4 Day 7Day 11 CuSO₄ (μg/ml) 125 7 10 15 37.5 13 16 18 12.5 15 17 18 Cu#16(μg/ml) 125 NG NG NG 37.5 8 11 12 12.5 11 14 14 Control 15 17 18 NGindicates no growth as assessed by phase microscopy at 40Xmagnification.4. Table 10 shows that both Cu# compositions which both containphosphorous acid were more effective than copper sulfate at inhibitingthe growth of Pm. Composition Cu#16 was made with ammonium hydrogencarbonate whilst Cu#27 contains ammonium hydroxide and as shown before(Table 6), this made no significant difference to the anti-fungalactivity of the composition. However, precipitation was noted incomposition Cu#16 after a few days at room temperature whilst this wasnot seen with Cu#27, suggesting that ammonium hydroxide may besurprisingly advantageous in the preparation of copper-ammoniumcomplexes when phosphorous acid is used to solubilize the complex.Compositions Cu—Zn#7 and Cu—Zn#10, which were equally effectiveinhibitors of Pm growth, differed in the use of ammonium carbonate andammonium hydroxide, respectively, to create the Cu—Zn-ammonium complexeswhich were solubilized with phosphorous acid in each case (Table 2).However, no precipitation was noted when ammonium carbonate was used toprepare Cu—Zn-ammonium complexes that were solubilized with phosphorousacid. Surprisingly, the Cu—Zn# compositions were more effectiveinhibitors of Pm growth than the Cu# compositions in terms of elementalcopper content. Thus, at a 1 percent dilution (150 micrograms/milliliterof copper) the Cu—Zn# compositions completely inhibited growth as didthe Cu# compositions—but at 375 micrograms/milliliter of elementalcopper. At a 0.3 percent dilution (45 micrograms/milliliter of copper)the Cu—Zn# compositions still strongly inhibited Pm growth, whilst at37.5 micrograms/milliliter the Cu# compositions were only slightlyinhibitory to Pm growth. Calculations indicate that the concentrationrequired to inhibit the growth of Pm by 50% was around 40micrograms/milliliter for the Cu—Zn# compositions and around 170micrograms/milliliter for the Cu# compositions. Therefore, these resultsshow that the Cu—Zn# compositions solubilized with phosphorous acid aresurprisingly more effective than the Cu# compositions solubilized withphosphorous acid at inhibiting the growth of Pm, regardless of theammonium salt used to form the complexes.

TABLE 10 The effects of copper sulfate, two Cu# compositions and twoCu—Zn# compositions on the growth of Pm on PDA after 3 days of culture.The Cu—Zn# compositions were used as a percent dilution of the stocksolutions (Table 2), so a 1% solution contains 150 μg/ml of elementalcopper (and zinc). Diameter of fungal growth (mm) Composition 12.5 μg/ml37.5 μg/ml 125 μg/ml 375 μg/ml CuSO₄ 17 17 13 3* Cu#16 17 15 10 NG Cu#2717 16 10 NG 0.03% 0.1% 0.3% 1% Cu—Zn#7 17 14  4 NG Cu—Zn#10 17 14  3* NGControl 17 NG indicates no growth as assessed by phase microscopy at 40Xmagnification. *Slight growth observed by phase microscopy at 40Xmagnification.

C. fraxinea is the cause of ash tree die-back which is a seriousforestry problem in Europe. A. niger causes black mold on certain fruitsand vegetables such as grapes, onions, and peanuts, and is a commoncontaminant of food. Fungi of the Genus Blumeria are widespread plantpathogens that cause powdery mildew on the leaves of many plantsincluding grapevines, Curcubits, onions, apple and pear trees. Theresults described above show that copper-ammonium andcopper-zinc-ammonium complexes solubilized with phosphorous acid arehighly effective at inhibiting the growth of the various plantpathogenic fungi tested and therefore may have practical applications inthe agricultural field for protecting of crops, plants, trees, flowersand ornamental shrubs. Surprisingly. Cu—Zn#7 and #10 were significantlymore effective than Cu#16 and #27 at inhibiting the growth of Powderymildew suggesting these compositions may have advantageous anti-fungalactivity against certain plant pathogens.

Example 5 Anti-Fungal/Yeast Assays with the Human Pathogens Candidaalbicans and Cryptococcus neoformans

The three strains of Candida albicans (C. albicans) used: 1-1, 3-1 and4-1 were isolated from human blood, vagina and oropharynx, respectively.Cryptococcus neoformans (C. neoformans) strain CCTP13 was used. Allstrains were cultured in RPMI-1640 medium at 37° C. The copper sulfateand Cu# compositions started at a high concentration of 200micrograms/milliliter, whilst the Cu—Zn# compositions started at a highconcentrations of 1% of the stock solution (Table 2) and zinc sulfateand phosphorous acid started with a high concentrations equivalent tothose in Cu—Zn#7 and #10 (see the legend of Table 1 for details)doubling dilutions (100 microliters) of the compositions were preparedin RPMI-1640 in a 96-well tissue culture plate. Overnight C. albicansand C. neoformans cultures were diluted 1:40 and 1:10 respectively withfresh RPMI containing 40 mM MOPS buffer and 90 microliters was added tothe compositions in the plate. Resazurin A (10 microliters of a 0.0675%wt/vol solution in distilled water) was added as an indicator of growth,and the cultures were incubated at 37° C. for 24 hr. The minimuminhibitory concentration (MIC) of the compounds/compositions was definedas the lowest concentration at which the resazurin A indicator remainedblue (living cells convert the blue dye to a pink color).

Results

Table 11 shows that all of the strains tested were surprisinglysensitive to copper, with MICs similar to those seen with MRSA and A.baumannii (around 6 micrograms/milliliter; Table 6), whether in the formof copper sulfate or the acid-solubilized copper-ammonium compositions(Cu#).

The acid-solubilized Cu—Zn-ammonium complex compositions #1 and #7 whichcontain equimolar amounts of elemental copper and zinc with hydrochloricand phosphorous acids, respectively, were slightly more effectiveinhibitors of C. albicans growth than copper sulfate and the Cu#compositions in terms of copper ion concentration (MIC=4.7micrograms/milliliter of elemental Cu²⁺). The yeast C. neoformans wasalso highly sensitive to copper regardless of the form, but was lesssensitive to Cu—Zn#1 than the C. albicans strains. The growth of the C.albicans strains was not affected by zinc sulfate or phosphorous acid(MIC>150 and >500 micrograms/milliliter, respectively). The growth of C.neoformans was also unaffected by zinc sulfate.

TABLE 11 The effect of various compositions on the growth of three C.albicans strains and a strain of C. neoformans. MIC (μg/ml Cu²⁺)Composition 1-1 3-1 4-1 C. neoformans CuSO₄ 6.3 6.3 6.3 6.3 Cu#3 6.3 6.36.3 6.3 Cu#9 6.3 6.3 6.3 6.3 Cu#12 6.3 6.3 6.3 6.3 Cu#16 6.3 6.3 6.3 NTCu—Zn#1  4.7* 4.7 4.7 18.8  Cu—Zn#7 4.7 4.7 4.7 NTZnSO₄ >150 >150 >150 >150    H₃PO₃ >500 >500 >500 NT *The Cu—Zn#compositions contain 4.7 μg/ml of both Cu²⁺ and Zn²⁺. **A 1% vol/volsolution of the Cu—Zn#7 and #10 stock compositions (see Table 2)contains 150 μg/ml of zinc sulfate and 500 μg/ml of phosphorous acid. NT= not tested.

These results show that C. albicans strains isolated from a variety ofdifferent anatomical sites are all highly sensitive to the presence ofcopper when grown in RPMI medium. Thus, it may be concluded that eitherof the Cu# or Cu—Zn# compositions may be useful in the topical treatmentof C. albicans-related diseases such as vaginal and oral thrush.

C. neoformans was also very sensitive to copper sulfate and the Cu#compositions, but was much less sensitive to Cu—Zn#1 and zinc sulfate.C. neoformans causes severe meningitis in people with HIV/AIDS andselective delivery of a copper-containing complex to the brain may be apotential therapy for this lethal disease.

Example 6 Stability Tests

Samples (3 milliliters) of composition Cu#1 and Aloe vera gel withpreservatives containing 1% vol/vol of composition Cu#1 were placed in 7milliliter sterile plastic tubes and replicate samples were stored underfour conditions; at 37° C. in a humidified incubator, at roomtemperature (around 22° C.) in a cupboard, at 4° C. in a cold room, andat −20° C. in a freezer. After 5 months of storage, a set of samples ofCu#1 and the gel containing 1% Cu#1 were allowed to come to roomtemperature. The gel samples (5 microliters) and the samples of Cu#1(diluted 1:1 or 1:10 vol/vol in distilled water and 5 microliters placedon a paper disc) were tested against bacterial strain S1 (S. aureus) inthe agar plate test, and after 24 hour culture at 37° C., the diametersof the zones of inhibition were measured as described in EXAMPLE 1.

Results

The samples of the gel containing 1% of composition Cu#1 all gave zonesof inhibition of 6 millimeters against S1 (S. aureus), indicating thatstorage at a wide range of temperatures had no effect on theanti-bacterial activity of the gels.

Table 12 shows the zones of inhibition for the samples of compositionCu#1 stored at various temperatures for 5 months. The sample stored at37° C. was slightly more inhibitory to the growth of S1 (S. aureus) thanthe other samples which gave very similar zones of inhibition at bothconcentrations tested. These results show that composition Cu#1 retainsits antibacterial activity after 5 months of storage at a wide range oftemperatures.

TABLE 12 The effect of samples of composition Cu#1 stored at varioustemperatures for 5 months on the growth of bacterial strain S1 (S.aureus) in the agar plate test. Zone of inhibition (mm) Cu#1 storagetemperature 1:1 dilution 1:10 dilution  37° C. 23 12 Room temperature(~22° C.) 19 10  4° C. 20 9 −20° C. 18 8

While the various objects of this invention have been described inconjunction with preferred embodiments thereof, it is evident that manyalternatives, modifications, and variations will be apparent to thoseskilled in the art. Accordingly, it is intended to embrace all suchalternatives, modifications and variations that fall within the spiritand broad scope of this specification, claims and the attached drawings.

What is claimed is:
 1. An antimicrobial soluble copper compositioncomprising: a solution comprising 10-160 grams/liter hydrated coppersulfate; a basic ammonium salt added to the hydrated copper sulfatesolution at 15-60 grams/liter to generate an insoluble copper-ammoniumcomplex; and at least one water soluble acid selected from the groupconsisting of phosphorous acid and phosphoric acid added to the hydratedcopper sulfate solution to solubilize the copper-ammonium complex and tocontrol the pH of the clear blue acid-solubilized copper-ammoniumsolution thus formed; wherein when present the amount of phosphorousacid is 24-80 grams/liter and wherein when present the amount ofphosphoric acid is 17-85 grams/liter; the resulting composition havingsynergistic antimicrobial properties greater than the antimicrobialproperties of each constituent component.
 2. The antimicrobialcomposition of claim 1, wherein the basic ammonium salt is selected fromthe group consisting of ammonium hydroxide, ammonium carbonate, andammonium hydrogen carbonate.
 3. The antimicrobial composition of claim1, wherein the water is selected from the group consisting of distilledwater, deionized water, purified water, filtered water, pharmaceuticalgrade water, medical grade water, and reverse osmosis water.
 4. Theantimicrobial composition of claim 1 wherein the hydrated copper sulfatesolution further comprises 11-66 grams/liter of a zinc salt in solutionforming a copper-zinc-ammonium complex upon the addition of the basicammonium salt.
 5. The antimicrobial composition of claim 4 wherein thezinc salt is selected from the group consisting of anhydrous zincsulfate, anhydrous zinc chloride, hydrated zinc sulfate, and hydratedzinc chloride.
 6. The antimicrobial composition of claim 4 wherein theammonium salt is dissolved in water.
 7. The antimicrobial composition ofclaim 1 further comprising a carrier selected from the group consistingof a gel, an ointment, an oil, a paste, a medicament, a sprayablesolution, a dressing solution, an irrigation solution, a cream, a soap,a detergent, a wash, and a foam.
 8. The antimicrobial composition ofclaim 1, wherein said antimicrobial composition is a plant protectionproduct.
 9. An antimicrobial composition produced by the process of:combining 10-160 grams/liter of hydrated copper sulfate with water;stirring the combined hydrated copper sulfate and water mixture; adding15-60 grams/liter of a basic ammonium salt to the resulting mixture togenerate an insoluble copper-ammonium complex; and adding a watersoluble acid selected from the group consisting of phosphorous acid andphosphoric acid to the copper-ammonium complex to solubilize thecopper-ammonium complex and to control the pH of the clear blue solutionthus formed; wherein when present the amount of phosphorous acid is24-80 grams/liter and wherein when present the amount of phosphoric acidis 17-85 grams/liter, the resulting composition having synergisticantimicrobial properties greater than the antimicrobial properties ofeach constituent component.
 10. The antimicrobial composition of claim9, wherein the water is selected from the group consisting of distilledwater, deionized water, purified water, filtered water, pharmaceuticalgrade water, medical grade water, and reverse osmosis water.
 11. Theantimicrobial composition of claim 9 wherein the basic ammonium salt isselected from the group consisting of ammonium hydroxide, ammoniumcarbonate, and ammonium hydrogen carbonate.
 12. The antimicrobialcomposition of claim 11 wherein the hydrated copper sulfate solutionfurther comprises 11-66 grams/liter of a zinc salt in solution forming acopper-zinc-ammonium complex upon the addition of the ammoniumhydroxide, ammonium carbonate or ammonium hydrogen carbonate.
 13. Theantimicrobial composition of claim 9 wherein the zinc salt is selectedfrom the group consisting of anhydrous zinc sulfate, anhydrous zincchloride, hydrated zinc sulfate, and hydrated zinc chloride.
 14. Theantimicrobial composition of claim 9 wherein the basic ammonium salt isdissolved in water before being combined with the hydrated coppersulfate and water mixture.
 15. The antimicrobial composition of claim 9further comprising a carrier selected from the group consisting of agel, an ointment, an oil, a paste, a medicament, a sprayable solution, adressing solution, an irrigation solution, a cream, a soap, a detergent,a wash, and a foam.
 16. A method of producing an antimicrobialcomposition comprising the steps of: combining 10-160 grams/liter ofhydrated copper sulfate with water; stirring the combined hydratedcopper sulfate and water mixture; adding 15-60 grams/liter of a basicammonium salt to the resulting mixture to generate an insolublecopper-ammonium complex; and adding a water soluble acid selected fromthe group consisting of phosphorous acid and phosphoric acid to thecopper-ammonium complex to solubilize the copper-ammonium complex and tocontrol the pH of the clear blue antimicrobial composition thus formed;wherein when present the amount of phosphorous acid is 24-80 grams/literand wherein when present the amount of phosphoric acid is 17-85grams/liter; the resulting composition having synergistic antimicrobialproperties greater than the antimicrobial properties of each constituentcomponent.